Gα Signaling Is Associated with Sexual Dimorphic Expression of the Clock

www.oncotarget.com Oncotarget, 2018, Vol. 9, (No. 54), pp: 30213-30224

Research Paper: Pathology

Gαi3 signaling is associated with sexual dimorphic expression of the clock-controlled output gene Dbp in murine liver

Madhurendra Singh1,8, Laura Bergmann1, Alexander Lang1, Katja Pexa1, Fabian Kuck1, Dennis Stibane1, Linda Janke5, Hakima Ezzahoini1, Antje Lindecke2, Constanze Wiek3, Helmut Hanenberg3,7, Karl Köhrer2, Charlotte von Gall4, Hans Reinke5,6 and Roland P. Piekorz1 1Institut für Biochemie und Molekularbiologie II, Medizinische Fakultät der Heinrich-Heine-Universität, Düsseldorf, Germany 2Biologisch-Medizinisches Forschungszentrum (BMFZ), Medizinische Fakultät der Heinrich-Heine-Universität, Düsseldorf, Germany 3Hals-Nasen-Ohren-Klinik, Medizinische Fakultät der Heinrich-Heine-Universität, Düsseldorf, Germany 4Institut für Anatomie II, Medizinische Fakultät der Heinrich-Heine-Universität, Düsseldorf, Germany 5Institut für Klinische Chemie und Laboratoriumsmedizin, Medizinische Fakultät der Heinrich-Heine-Universität, Düsseldorf, Germany 6IUF – Leibniz Institut für Umweltmedizinische Forschung, Düsseldorf, Germany 7Klinik für Kinderheilkunde III, Universitätsklinikum Essen, Universität Duisburg-Essen, Essen, Germany 8Current address: Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden Correspondence to: Roland P. Piekorz, email: [email protected] Keywords: circadian regulation; galphai3/GNAI3; CREB; cytochrome P450; albumin D-box binding protein; Pathology Received: February 07, 2017 Accepted: June 14, 2018 Published: July 13, 2018 Copyright: Singh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

The albumin D-box binding protein (DBP) is a member of the PAR bZip (proline and acidic amino acid-rich basic leucine zipper) transcription factor family and functions as important regulator of circadian core and output gene expression. Gene expression of DBP itself is under the control of E-box-dependent binding by the Bmal1-Clock heterodimer and CRE-dependent binding by the cAMP responsive element binding protein (CREB). However, the signaling mechanism mediating CREB- dependent regulation of DBP expression in the peripheral clock remains elusive. In this

study, we examined the role of the GPCR (G-protein-coupled receptor)/Gαi3 (Galphai3) controlled cAMP-CREB signaling pathway in the regulation of hepatic expression of core clock and clock-regulated genes, including Dbp. Analysis of circadian gene expression revealed that rhythmicity of hepatic transcript levels of the majority of core

clock (including Per1) and clock-regulated genes were not affected by Gαi3 deficiency.

Consistently, the period length of primary Gαi3 deficient tail fibroblasts expressing

a Bmal1-Luciferase reporter was not affected. Interestingly, however, Gαi3 deficient female but not male mice showed a tendentiously increased activation of CREB (nuclear pSer133-CREB) accompanied by an advanced peak in Dbp gene expression

and elevated mRNA levels of the cytochrome P450 family member Cyp3a11, a target gene of DBP. Accordingly, selective inhibition of CREB led to a strongly decreased expression of DBP and CYP3A4 (human Cyp3a11 homologue) in HepG2 liver cells.

In summary, our data suggest that the Gαi3-pCREB signalling pathway functions as a regulator of sexual-dimorphic expression of DBP and its xenobiotic target enzymes Cyp3a11/CYP3A4.

www.oncotarget.com 30213 Oncotarget Introduction cell- and tissue-type specific expression profiles. Overall,

Gαi2 represents the predominantly and ubiquitously

The circadian clock system in mammals is expressed isoform, whereas Gαi3 is found in all peripheral hierarchically organized comprising a central master tissues with only low detectable expression in neuronal

clock and peripheral clocks [1, 2]. The central clock cells [28–30]. Of note, both Gαi2 and Gαi3 are expressed

localizes to the suprachiasmatic nuclei (SCN) of the in the liver where Gαi3 exhibits an isoform-specific anterior hypothalamus and is mainly synchronized by crucial function in the anti-autophagic action of insulin environmental light [3, 4]. In contrast, synchronization of in hepatocytes [29]. However, the function of pertussis

peripheral clocks, which are present in nearly all tissues toxin-sensitive Gi proteins in circadian signaling and gene and organs, occurs by hormonal and neuronal cues and/ expression is largely unclear.

or food intake [5–7]. In central and peripheral clocks, Here, we analyzed the role of Gαi3 in rhythmic the molecular clockwork is composed of intertwined pSer133-CREB activation and clock gene expression in positive and negative feedback loops at the transcriptional the liver in mice. The phase of clock genes Per1, Per2, and (post)translational level [4, 8–10]. In particular, Bmal1, Rev-Erba, Cry1, and Cry2 as well as clock output −/− core molecular clockwork components encoded by so- genes Hlf and Tef was not different between Gαi3 and called clock genes regulate positively (Clock, Bmal1) or wild-type littermates of both genders. In contrast, in −/− negatively (Cry1/Cry2 and Per1/Per2) rhythmic expression Gαi3 females the phase of the clock output gene DBP of their target genes (i.e., clock output genes) through was advanced. This was associated with a slight increase in transcriptional control via so-called E-box elements pCREB and DBP protein levels in females. Moreover, the [11]. Important clock output genes include members of expression level of the DBP target gene and cytochrome the family of PAR-domain basic leucine zipper (PAR P450 family member Cyp3a11 were significantly higher bZip) transcription factors, i.e. hepatic leukemia factor in females at mid-day and mid-night, and in males at early

(HLF), thyrotrophic embryonic factor (TEF), and albumin day. Thus, our data indicate that the Gαi3-pCREB signaling D-box binding protein (DBP) [12]. The latter activates pathway functions as a novel direct (via DNA binding) or gene expression of Per1/2 via DBP binding sites (so- indirect regulator of sexual-dimorphic hepatic expression called D-box elements) therefore modulating the levels of DBP and its xenobiotic targets. of core clock gene products [13, 14]. Moreover, PAR bZip proteins control the circadian expression of hepatic Results enzymes and regulators involved in endobiotic and xenobiotic biotransformation and drug metabolism [15], Rhythmic clock gene expression is not affected including isoenzymes of the cytochrome P450 family of by Gα deficiency monooxygenases. i3 Several findings indicate an important role of the The cAMP-CREB signaling pathway, that is

Gs/Gi-protein-controlled cAMP-CREB signaling pathway under the control of heterotrimeric Gs and Gi proteins, in the regulation of clock gene expression. Treatment of regulates the expression of core clock and clock-regulated Rat-1 fibroblasts with forskolin, which activates adenylyl genes, including Per1 and DBP [16, 17]. To characterize

cyclase and therefore increases intracellular cAMP the role of the Gαi isoform Gαi3 in this pathway, Gαi3 concentrations, resulted in elevated pSer133-CREB levels deficient mice [29] and wild-type control animals were concomitant with an enhanced circadian accumulation of kept in a 12 h light/12 h dark cycle setting and liver DBP, Per1, and Per2 [16]. Moreover, the cAMP-CREB tissue was collected every 6 h for up to 24 h followed signaling pathway positively regulates Per1 expression in by gene expression analysis via quantitative real-time human hepatoma cells [17] as well as the light-induced polymerase chain reaction (qRT-PCR). As depicted Per1 expression in the SCN [18, 19]. In the SCN, CREB is in Figure 1, the phases of rhythms in transcript levels rapidly phosphorylated at Ser133 after light at night [20– of the core molecular clockwork components (Per1, 22], but circadian oscillation of intracellular cAMP is also Per2, Bmal1, Rev-erba, Cry1, and Cry2) and clock- important for intercellular coupling of SCN neurons [23]. regulated genes (Hlf and Tef) and their expression levels −/− In liver, intracellular cAMP concentrations and CREB were comparable between Gαi3 mice and wild-type −/− activation are modulated by Cry1 [24]. controls of both genders. In male Gαi3 mice, the peak Signaling by GPCRs via heterotrimeric Gs (i.e., in Cry1 expression at ZT18 was significantly higher as

Gαsβγ) and Gi (i.e., Gαiβγ) proteins activates or inhibits, compared to wild-type controls (Figure 1B). To address respectively, cAMP production by adenylyl cyclases, these molecular findings at a functional level, we next −/− thereby regulating downstream cAMP-PKA (protein isolated primary tail fibroblasts from wild-type and Gαi3 kinase A)-CREB signaling [25]. The Gαi subunits female mice followed by lentiviral transduction of a

comprise three highly homologous isoforms, Gαi1, Gαi2, Bmal1 promoter-driven luciferase reporter construct and

and Gαi3 [26, 27], which display a high amino acid subsequent real-time bioluminescence monitoring. As sequence identity together with overlapping as well as indicated in Supplementary Figure 1A, and consistent with www.oncotarget.com 30214 Oncotarget the expression profiling results in Figure 1, recording of to normalize gene expression analysis. This phase-shifted luciferase activity revealed a circadian period length that expression was associated with increased DBP protein −/− was nearly identical between Gαi3 deficient fibroblasts levels in Gαi3 female vs. wild-type livers during the and wild-type cells (25.1 vs. 25.0 h, respectively; dark phase (ZT12 and ZT18) (Figure 2C). In contrast, the Supplementary Figure 1B). rhythm in hepatic expression of DBP in male mice was comparable in both genotypes at the mRNA (Figure 2B and Supplementary Figure 2B) and protein level (Figure Gαi3 deficient female mice exhibit a phase-shifted rhythm of hepatic DBP expression 2D). DBP mRNA levels in male mice showed the same peak as wild-type females peaking at ZT12 similarly as Interestingly, in contrast to the clock output genes previously described for C57Bl/6 wild-type mice [15]. and PAR bZip members Tef and Hlf, whose transcript The sexual dimorphic, phase-shifted expression of DBP

levels were apparently not influenced by Gαi3 deficiency became particularly evident when the data from Figure 2A, (Figure 1A), rhythmic expression of the PAR bZip B were compared between the wild-type (Supplementary −/− member DBP was phase advanced by six hours in Gαi3 Figure 3A) and Gαi3 knockout (Supplementary Figure females as compared to wild-type females (Figure 2A 3B) of both sexes. In contrast, an analogous re-analysis and Supplementary Figure 2A). Here, comparable results of the data from Figure 1 revealed rather comparable were obtained using Gapdh, β-Actin, or EF1a as controls circadian rhythms in the transcript levels of core molecular

-/- Figure 1: Rhythmic expression of core clock genes and clock-regulated genes in the liver of Gαi3 mice. Quantitative real-time PCR analysis of rhythmic expression of core clock genes and clock output genes in the liver of female (A) and male (B) Gαi3 deficient mice vs. wild-type control animals. Mice were sacrificed and analyzed every six hours at the indicated time points (ZT 0 to ZT 18). Transcript levels of the indicated genes were normalized to the endogenous control Gapdh. Shown are 2-ΔCt values. Results are expressed as mean ± s.d. of six animals analyzed per genotype and time point (*p < 0.05; **p < 0.01 as compared to corresponding wild- type control animals).

www.oncotarget.com 30215 Oncotarget clockwork components (with the exception of Per1 and important regulator of hepatic expression of core clock Per2 which displayed slightly to significantly higher and clock-regulated genes [16, 17, 19]. Initial analysis transcript levels in females as compared to males of both of total cell lysates of liver tissue indicated that the genotypes) and other clock-regulated genes (like Hlf and cellular levels of pCREB (pSer133-CREB) were higher −/− Tef) (Supplementary Figure 4). Of note, Gαi3 in the liver in Gαi3 female mice as compared to corresponding per se showed no rhythmic expression at the mRNA level wild-type female controls (Supplementary Figure 6A). (data now shown) or the protein level (Supplementary In contrast, total pCREB levels were rather comparable −/− Figure 5). The latter is consistent with a lack of temporal between male Gαi3 and wild-type mice (as judged by

changes of Gαi amounts in the murine SCN [23]. Taken pCREB/CREB signal ratios) thereby following a similar together, hepatic expression of the PAR bZip member circadian rhythm (Supplementary Figure 6B). Given DBP in female mice seems to underlie a sexual-dimorphic that pSer133-CREB localizes in the nucleus thereby

regulation that is dependent on Gαi3. functioning as transcription factor [31], we next analyzed nuclear extracts isolated from liver tissue. Consistent −/− Evidence for increased circadian cAMP-CREB with the findings above, Gαi3 female mice displayed slightly higher nuclear levels of pCREB as compared to signaling in the liver of Gαi3 deficient female mice wild-type females (Figure 3A, 3B), whereas there was no obvious difference in nuclear pCREB levels between −/− We next addressed the role of Gαi3 in the GPCR/ male Gαi3 and wild-type mice (Supplementary Figure 7).

Gαi controlled cAMP-CREB signaling pathway as an These findings indicate a sexual-dimorphic inhibitory

-/- Figure 2: Rhythmic expression of DBP in the liver of female Gαi3 mice is phase advanced (A, B) Quantitative real-time PCR analysis of

rhythmic expression of DBP mRNA in the liver of female (A) and male (B) Gαi3 deficient mice as compared to wild-type control animals. Dbp transcript levels were normalized to the endogenous control Gapdh. Shown are 2-ΔCt values. (C, D) Representative immunoblots of

rhythmic expression of the DBP protein in the liver of female (C) and male (D) Gαi3-deficient mice and wild-type controls. GAPDH and β-Actin were employed as loading controls. Relative protein levels (DBP/GAPDH) were determined by densitometric analysis using ImageJ software (lower panels in C and D). Mice were sacrificed and analyzed every six hours at the indicated time points (ZT 0 to ZT 18). Results are expressed as mean ± s.d. of six animals (mRNA) or four animals (immunoblot) analyzed per genotype and time point (*p < 0.05; **p < 0.01 as compared to corresponding wild-type control animals). www.oncotarget.com 30216 Oncotarget impact of Gαi3 on the cAMP-CREB pathway and suggest CYP3A4 in human HepG2 cells upon selective inhibition

that the Gαi3-pCREB axis may function as a candidate of CREB (Supplementary Figures 9 and 10). determinant of female specific expression of DBP. The important role of CREB is emphasized by the observation Discussion that selective inhibition of CREB led to a clear decrease in the expression of DBP (Supplementary Figures 9 and 10). This study provides novel insight into the role of

the heterotrimeric G protein Gαi3 as upstream regulator of Elevated transcript levels of the DBP cytochrome the cAMP-pCREB signalling pathway in rhythmic gene

P450 target gene Cyp3a11 in Gαi3 deficient expression in the liver. Our data indicate (i) that gene female mice ablation of Gαi3 in mice has only modest effects on overall core clock and clock output gene expression and does DBP drives transcriptionally the rhythmic not affect the period length of clock gene expression; (ii) expression of the xeno- and endobiotic target genes interestingly, slightly increased levels of nuclear activated CYP2A4 (Steroid 15α-hydroxylase), CYP2A5 (Coumarin CREB (pSer133-CREB) which are linked to a phase- 7-hydroxylase), and CYP3A4 (Cyp3a11 in mouse) [32], shifted and increased expression of its potential target which are involved in cholesterol/steroid metabolism gene and PAR bZip transcription factor DBP is detectable or biotransformation of lipophilic xenobiotics. In Gα i3 in livers from Gαi3 deficient female mice; (iii) accordingly, deficient females, transcript levels of Cyp3a11 at ZT06 the DBP target gene and cytochrome P450 family member and ZT18 were significantly higher as compared to levels Cyp3a11 is found at higher transcript levels in the liver in the corresponding wild-type females (Figure 4A). −/− of Gαi3 female mice. Thus, Gαi3 may function as novel In comparison, Gαi3 deficient males displayed levels regulator of sexual-dimorphic, cAMP-PKA-pCREB of Cyp3a11 that were significantly higher at ZT0 as driven expression of DBP together with its xenobiotic compared to corresponding wild-type males (Figure 4B). target genes in the liver (Figure 5). Of note, selective In contrast, levels of CYP2A4/CYP2A5 were not different inhibition of CREB led to a clearly decreased expression

between Gαi3 deficient and wild-type mice of both genders of DBP and CYP3A4 (human Cyp3a11 homologue) in

(Figure 4A, 4B). Taken together, upon Gαi3 deficiency, HepG2 hepatoma cells (Supplementary Figures 9 and increased signaling by the cAMP-pCREB axis seems to 10) further stressing the downstream role of CREB in the translate into higher hepatic Cyp3a11 gene expression regulation of DBP and its target genes. that was more pronounced in female mice and potentially The phenotypic characterization of Gαi deficient dependent on transcriptional regulation via DBP. The mouse lines has been expanded in the recent years important role of CREB signaling is emphasized by a revealing that Gαi2 and Gαi3 take over gene-specific as clear decrease in the expression of DBP and its target gene well as shared physiological functions in various organs

-/- Figure 3: Gαi3 female mice display increased nuclear pSer133-CREB levels in the liver. (A) Immunoblot analysis of -/- pSer133-CREB and CREB protein levels in nuclear extracts from livers of female wild-type vs. Gαi3 mice. (B) Quantitative analysis of relative protein levels (pSer133-CREB/CREB) was performed by ImageJ software. Results are expressed as mean ± s.d. of three independent experiments. Mice were sacrificed and analyzed every six hours at the indicated time points (ZT 0 to ZT 18). ATF1, activating transcription factor 1. www.oncotarget.com 30217 Oncotarget and cell types in-vivo [28, 29, 33–41]. The liver expresses circadian gene expression in the SCN and an altered

both Gαi2 and Gαi3 with the latter exhibiting an isoform- circadian rhythmicity [43, 44]. Here, although the identity specific and crucial function in the anti-autophagic action of the involved GPCRs in the regulation of circadian

of insulin in hepatocytes [29]. Although a comparative signaling is currently only speculative, the Gi/Gq-coupled characterization of the hepatic expression of core clock melatonin receptors MT1 and MT2 [45] represent prime

and clock-regulated genes in Gαi2 knockout mice is not candidates given that they play an important role in

yet available, we provide evidence that Gαi3 deficiency central clock synchronization. MT1 and MT2 are both per se alters expression of DBP and its target genes in a expressed in the liver [46] where they display circadian

Gαi isform-specific manner apparently via a desinhibited rhythmic changes in gene expression [47]. Interestingly, cAMP-pCREB pathway. and consistent with our findings in Figure 2, DBP mRNA Several observations emphasize a critical role of levels are phase-advanced in mice lacking either MT1,

GPCR/Gs (cAMP↑) and GPCR/Gi (cAMP↓) pathways in MT2, or both receptors [46, 48], indicating that these the regulation of rhythmic gene expression. Interestingly, GPCRs are involved in the regulation of DBP expression.

Cry1 interacts directly with Gas at the GPCR level and However, since the mouse lines analyzed in this study thereby inhibits adenylyl cyclase activity and accumulation were on the melatonin-deficient C57Bl/6 genetic of cAMP and activation of CREB [24]. These findings background [49], backcrossing with mice on the melatonin provided an explanation how the rhythmic expression proficient C3H background is a prerequisite to ultimately of Cry1 translates directly into a circadian regulation determine changes in melatonin receptor signaling and

of cAMP signaling in hepatic glucose metabolism, in rhythmic hepatic gene expression upon Gαi3 deficiency. particular gluconeogenesis [24]. Moreover, functional In terms of the regulation of DBP expression,

inactivation of Gi-proteins by pertussis toxin [42] or a previous work showed that maximal and minimal levels reduction of cAMP levels by MDL-12330A mediated of DBP mRNA occur approximately four hours later in inhibition of adenylyl cyclase led to a desynchronized the liver as compared to the SCN [50], suggesting that

Figure 4: Transcript levels of the DBP target gene and cytochrome P450 family member Cyp3a11 are increased in the -/- liver of Gαi3 female mice. Quantitative real-time PCR analysis of rhythmic expression of Cyp3a11 and Cyp2a4/a5 genes in the liver of female (A) and male (B) Gαi3 deficient mice vs. wild-type control animals. Mice were sacrificed and analyzed every six hours at the indicated time points (ZT 0 to ZT 18). Transcript levels of Cyp3a11 and Cyp2a4/a5 were normalized to the endogenous control Gapdh. Shown are 2-ΔCt values. Results are expressed as mean ± s.d. of five animals analyzed per genotype and time point (*p < 0.05; **p < 0.01 as compared to corresponding wild-type control animals). www.oncotarget.com 30218 Oncotarget additional regulatory mechanisms (besides transcriptional however, we failed to detect any obvious differences in control via E-box elements) must be involved in the nuclear vs. cytoplasmic CLOCK protein levels between

control of rhythmic DBP expression in peripheral wild-type and Gαi3 deficient hepatocytes at ZT6 and ZT12 tissues when compared to the SCN [13, 51–54]. Indeed, (Supplementary Figure 11). treatment of Rat-1 fibroblasts with the adenylyl cyclase Increasing evidence argues for a role of sexual activator forskolin was sufficient to increase cAMP- dimorphism in life span regulation, disease sensitivity, pCREB signalling and trigger circadian expression of DBP and particularly drug metabolism in females vs. males [16, 55]. These findings indicate that candidate cAMP [3, 58–61]. The liver represents a bona fide sexual- inducible promoter elements (CRE) and pCREB binding dimorphic organ with a rhythmic physiology especially

sites [56], which are regulated by Gαi3-mediated signaling, in terms of its detoxification function [62, 63]. In these are present in the Dbp gene (“direct model”). Indeed, regards, indications for a sexual dimorphic signalling and besides the two E-box binding sites (5’-CACGTC-3’), expression of core clock and clock regulated genes in the two CRE half sites (5’-CGTCA-3’) can be detected in liver include based on our study (i) slightly to significantly the promoter region of the murine Dbp gene (Singh higher transcript levels of Per1 and Per2 in females vs. and Piekorz, unpublished). However, it remains to be males (Supplementary Figure 4), (ii) potential differences determined whether these sites function as pCREB in pCREB signalling given the levels and rhythms of binding elements. Alternatively, one could envision that pSer133-CREB in female vs. male animals (Supplementary the subcellular (nuclear vs. cytoplasmic) distribution of Figure 6; Figure 3 and Supplementary Figure 7), (iii) a BMAL-1/CLOCK, that regulates Dbp gene expression phase-shifted expression of DBP that became particularly through E-box binding [57], has become disturbed by evident when comparing wild-type (Supplementary Figure

Gαi3 deficiency in hepatocytes (“indirect model”). So far, 3A) and Gαi3 knockout (Supplementary Figure 3B) of both

Figure 5: Schematic model suggesting a putative role of the Gas/Gαi3-CREB signalling pathways in sexually-dimorphic regulation of DBP expression in female vs. male mouse liver. In females, loss of Gαi3 leads presumably via desinhibition of adenylyl cyclase and hence increased pSer133-CREB levels to a phase-advanced and increased expression of DBP in the liver, concomitant -/- with an increase in transcript levels of Cyp3a11, a putative DBP target gene. In Gαi3 males, this signalling pathway may be less active provided that an increased CRY1 expression (as indicated in Figure 1) results in a stronger inhibitory role of CRY1 on cAMP levels (via

Gs inhibition) [24]. B: BMAL1; C: CLOCK; CRE: cAMP responsive element; CREB, cAMP response element binding protein; CRY1, cryptochrome circadian clock 1; Cyp3a11: cytochrome P450, family 3, subfamily a, polypeptide 11; DBP: albumin D-box binding protein;

PKA: protein kinase A; Gi3 and Gs: heterotrimeric guanine nucleotide binding proteins which upon activation cause a decrease or increase in intracellular cAMP levels, respectively. www.oncotarget.com 30219 Oncotarget sexes, and (iv) a significantly higher circadian expression Isolation of liver tissue of CYP2A4/2A5 in females vs. males independent of the genotype (Supplementary Figure 8). As summarized in Experimental animals (male and female mice −/− between twelve to eighteen weeks of age) were maintained the model in Figure 5, in Gαi3 males, the cAMP-PKA- pCREB signalling pathway may be less active provided under standard conditions with 12 h light / 12 h dark that the increased CRY1 expression (Figure 1B) results cycles. Animals were killed by cervical dislocation and in a stronger inhibitory role of CRY1 on cAMP levels livers were obtained at the Zeitgeber times (ZT) 0, 6, 12, and 18 (ZT 0: Light ON; ZT 12: Light OFF) and (via Gs inhibition) essentially as suggested by Zhang et al. [24]. Of note, the increased CRY1 expression in immediately frozen in aliquots in liquid nitrogen for −/− further analysis. Gαi3 males could be due to a phase shift/earlier onset of Cry1 expression between ZT12 and ZT18, given that ZT intervals of six hours (instead of four hours) have been RNA isolation and quantitative real-time PCR analyzed in this study. (qRT-PCR)

CYP2A4/2A5 belongs to the cytochrome P450 family of monooxygenases, whose expression is under diurnal Total RNA from liver tissue was extracted using RNeasy mini kit spin columns (QIAGEN). RNA purity control by the circadian clock via PAR bZip proteins, and concentration was determined using a NanoDrop including DBP. These transcription factors also control the spectrophotometer. One µg of liver RNA was reverse- expression of additional enzymes and regulators involved transcribed using oligo(dT) primers and the ImProm™ in endobiotic and xenobiotic biotransformation and drug 15 II Reverse Transcription System (Promega) according to metabolism [15]. In particular, DBP per se is able to the manufacturer’s specifications. Relative quantification transcriptionally regulate the circadian accumulation of the of mRNA was carried out using quantitative real- target genes CYP2A4 (Steroid 15α-hydroxylase), CYP2A5 time PCR (qRT-PCR; 7500 Real-Time PCR System; (Coumarin 7-hydroxylase), and CYP3A4 (Cyp3a11 in Applied Biosystems) and specific TaqMan probes mouse [32]), given that these genes display D-box elements (Applied Biosystems) for core clock genes and clock in their promotor regions [64, 65]. CYP2A4/CYP2A5 is regulated genes [67]. Expression of target sequences was involved in cholesterol and bile acid metabolism [64], normalized to an endogenous control, glyceraldehyde-3- whereas CYP3A4/Cyp3a11 plays an important role in the phosphate dehydrogenase (GAPDH: Part No.: 4331182, metabolism of endogenous steroids and is responsible for Assay-ID: Mm99999915_g1). For further normalization biotransformation of approximately 50% of all prescription to other endogenous controls, β-Actin and EF1a were drugs [65, 66]. The profound role of PAR bZip family used (Schneider et al., 2014). qRT-PCR for cytochrome members especially in the regulation of drug metabolism P isoforms was performed using TaqMan probes for and biotransformation [15] becomes particularly obvious 450 Cyp3a11 (Part No.: 4331182, Assay-ID: Mm00731567_ in triple knockout mice lacking Dbp, Hlf, and Tef. These m1) and Cyp2a4/Cyp2a5 (Part No.: 4331182, Assay-ID: animals display a deregulated expression of numerous Mm00487248_g1). cytochrome P450 gene family members, an increased sensitivity to xenobiotics, and phenotypic alterations Total cell lysates and immunoblot analysis reminiscent of premature aging [12]. In summary, taking our findings in account it Total cell lysates from mouse liver were prepared remains to be particularly tested whether an increased as described [68] with minor modifications. Briefly, signaling by the cAMP-pCREB-DBP axis in the liver of snap-frozen liver tissue was homogenized with an Ultra- −/− Gαi3 female mice translates into an increased Cyp3a11 Turrax for 30–60 s at 4° C in 800 µl of lysis buffer activity and therefore improved biotransformation. (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1 mM EDTA; 1% Triton-X100; 0.5% deoxycholic acid; 1% SDS) Materials and Methods supplemented with cocktails of protease inhibitors (Roche) and phosphatase inhibitors (Sigma-Aldrich)]. Mice After sonication for 45 seconds, tissue homogenates were cleared by a centrifugation step (30.000 × g,

All experiments were performed with Gαi3-deficient 15 min). Lysates were subjected to protein electrophoresis mice on a C57Bl/6 background with corresponding employing 8.5–10% SDS-acrylamide gels or 6 M C57BL/6 wild-type animals used as controls. For breeding urea/SDS-PAGE gels and blotted onto nitrocellulose and maintenance, mice were kept under specific pathogen- membrane [29]. Membranes were blocked in 5% non- free conditions (SPF) with 12 h light / 12 h dark cycles and fat dry milk and incubated with primary antibodies at

free access to food and water at the local animal house 4° C overnight (anti-Gαi3, Santa Cruz sc-262, 1:1000; of the Heinrich-Heine-University Düsseldorf. The study anti-DBP, abcam ab22824, 1:1000; anti-phospho-CREB was performed in accordance with the national and local [pSer133-CREB], Cell Signaling 87G3 #9198, 1:1000; guidelines on animal care. anti-CREB, Cell Signaling 48H2 #9197, 1:1000; anti- www.oncotarget.com 30220 Oncotarget GAPDH, abcam ab8245, and anti-Actin C4, Millipore, Abbreviations both 1:5000). The anti-phospho-CREB antibody recognizes the phosphorylated form of CREB (pSer133-CREB) and ATF1: activating transcription factor 1; Cyp3a11:

the activating transcription factor-1 (ATF-1). Nuclear cytochrome P450: family 3: subfamily a: polypeptide 11; and cytoplasmatic CLOCK levels were detected using a CREB: cAMP responsive element binding protein; DBP:

primary antibody from Cell Signaling (D45B10, rabbit albumin D-box binding protein; Gαi: inhibitory G protein

mAb #5157; 1:1000). Antibodies against total Histone alpha subunit; Gi: heterotrimeric Gαiβγ protein; PAR bZip: (Cell Signaling #9715, 1:1000) or Actin (C4, Millipore, PAR-domain basic leucine zipper; PKA: protein kinase A; 1:5000) was employed to control protein loading of nuclear qRT-PCR: quantitative real-time PCR; ZT: Zeitgeber time. and cytoplasmatic fractions, respectively. Following three washing steps membranes were incubated with a ACKNOWLEDGMENTS AND FUNDING horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (Cell Signaling #7074) or a HRP-conjugated We thank Jutta Schröder for introduction into the polyclonal rabbit-anti-mouse IgG (Dako P0161) both at a ImageJ software and all current and former members of dilution of 1:5000 for 60 min at room temperature. Protein the institute for discussion and support. This work was signals were visualized by the ECL detection system (GE supported by the research commission of the medical Healthcare) and images were collected using an INTAS faculty of the Heinrich-Heine-University (grant 9772517), chemostar imager. the Deutsche Forschungsgemeinschaft (SFB 612, project A8), and the Deutsche Jose Carreras Leukämie-Stiftung Isolation of nuclear proteins from mouse liver e.V. (grant number DJCLS R15/10 to H.H.).

−/− Nuclear proteins from 12–18 weeks old Gαi3 Conflicts of interest and wild-type livers (ZT 0, 6, 12, and 18) were prepared essentially as described [69]. In brief, 0.5–0.6 g of liver The authors state no conflicts of interest. tissue was washed in phosphate buffered saline (PBS) buffer (pH 7.4) and disintegrated in 4 ml of ice-cold References

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